The role of sub-genomic RNA in discordant results from RT-PCR tests for COVID-19.

Reverse transcription-PCR (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when one RT-PCR target is positive for SARS-CoV-2 and one RT-PCR target is negative within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. It was hypothesized that concentration differences across targets may be due to the transcription of sub-genomic RNA, as this would result in an increase in the concentration of gene targets near the 3’ end of the SARS-CoV-2 genome. A panel of six digital droplet (dd)PCR assays was designed to quantitate the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity in such a way as to maximize gene-to-gene copy ratios. Eleven clinical nasopharyngeal swabs were also tested with this panel. In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. Both the highest specific infectivity (copies/pfu) and the highest differences between gene targets were observed at 6 hours post-infection (eclipse phase) in infected cells. The same trends in the relative abundance of copies across different targets observed in infected cells was observed in clinical samples, though trends were more pronounced in infected cells. This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by this data.

s-aligner: a greedy algorithm for non-greedy de novo genome assembly

Genome assembly is a fundamental tool for biological research. Particularly, in microbiology, where budgets per sample are often scarce, it can make the difference between an inconclusive result and a fully valid conclusion. Identifying new strains or estimating the relative abundance of quasi-species in a sample are some example tasks that can’t be properly accomplished without previously generating assemblies with little structure ambiguity and covering most of the genome. In this work, we present a new genome assembly tool based on a greedy strategy. We compare the results obtained applying this tool to the results obtained with previously existing software. We find that, when applied to viral studies, comparatively, the software we developed often gets far larger contigs and higher genome fraction coverage than previous software. We also find a significant advantage when applied to exceptionally large virus genomes.

460. Point-of-Care, In-Home SARS-CoV-2 IgG Antibody Testing to Assess Seroprevalence in At-Risk Health Care Workers

Background Health care workers are at significant risk for infection with the novel coronavirus SARS-CoV-2. Methods We utilized a point-of-care, lateral flow SARS-CoV-2 IgG immunoassay (RayBiotech) to conduct a seroprevalence study in a cohort of at-risk health care workers (n=339) and normal-risk controls (n=100) employed at an academic medical center. To minimize exposure risk while conducting the study, consents were performed electronically, tests were mailed and then self-administered at home using finger stick blood, and subjects uploaded a picture of the test result while answering an electronic questionnaire. We also validated the assay using de-identified serum samples from patients with PCR-proven SARS-CoV-2 infection. Results Between April 14th and May 6th 2020, 439 subjects were enrolled. Subjects were 68% female, 93% white, and most were physicians (38%) and nurses (27%). In addition, 37% had at least 1 respiratory symptom in the prior month, 34% had cared for a patient with known SARS-CoV-2 infection, 57% and 23% were worried about exposure at work or in the community, respectively, and 5 reported prior documented SARS-CoV-2 infection. On initial testing, 3 subjects had a positive IgG test, 336 had a negative test, and 87 had an inconclusive result. Of those with an inconclusive result who conducted a repeat test (85%), 96% had a negative result. All 3 positive IgG tests were in subjects reporting prior documented infection. Laboratory validation showed that of those with PCR-proven infection more than 13 days prior, 23/30 were IgG positive (76% sensitivity), whereas 1/26 with a negative prior PCR test were seropositive (95% specificity). Repeat longitudinal serologic testing every 30 days for up to 4 times is currently in progress. Conclusion We conducted a contact-free study in the setting of a pandemic to assess SARS-CoV-2 seroprevalence in an at-risk group of health care workers. The only subjects found to be IgG positive were those with prior documented infection, even though a substantial proportion of subjects reported significant potential occupational or community exposure and symptoms that were potentially compatible with SARS-COV-2 infection. Disclosures All Authors: No reported disclosures

1060 Screening For Obstructive Sleep Apnea In Severe Gastroesophageal Reflux Disease Patients: A Forgotten Association?

Introduction Obstructive sleep apnea (OSA) is an emerging epidemic in the USA and remains underdiagnosed. Investigations of Gastroesophageal reflux disease (GERD) poses a substantial burden on patient-welfare and costs to the health system. Current literature has highlighted the association between severe GERD and OSA, and other sleep disorders. We conducted a retrospective analysis of patient records undergoing Bravo pH monitoring for refractory GERD to measure the prevalence of OSA and screening. Methods Records of patients who underwent outpatient Bravo pH monitoring at a teaching hospital were reviewed from August 2018 to May 2019. 72 records were reviewed in our analysis. Analysis variables included age, gender, body mass index, history of hypertension and OSA. Outpatient records were reviewed for documentation for OSA or screening and demographics were obtained for calculation of a partial STOP-BANG score (a validated OSA screening tool). Results 8 out of 72 (11%) were excluded due to incomplete documentation regarding their history. Of the remaining 64, 2 had a known diagnosis of OSA (3%) and 1 was due a sleep study for maintenance insomnia. Of the remaining 61 patients, none had documentation of a history pertaining to sleep complaints or full screening for OSA. 4 of the 8 components to the STOP-BANG criteria were documented and used to measure risk of OSA in these patients. 23 (39%) patients had a score of 3 or above characterizing them as intermediate risk. The other 4 components were not used due to a lack of clinical information. 13 of these patients had a positive Bravo test, 2 had an inconclusive result and 8 had a negative result. Of the 61 patients in total, 31 had a positive Bravo result and 9 had an inconclusive result. Conclusion In our study, we found that 39% of patients based on demographic data were of intermediate risk of OSA. Over half of these patients had a positive result for GERD. Despite the increased awareness of sleep disorders it is still neglected despite prevalent associated comorbid conditions. This study highlights the failure to screen for this modifiable risk factor within a teaching environment. Support None

The US CDC RT-PCR probes for the Wuhan 2019-nCoV are non-specific for SARS - there is a better non-specific 500bp option (21852-22427) within the spike-protein

The World Health Organization has declared the Wuhan coronavirus [1–3] outbreak a public health emer- gency on 30th Jan, 2020. An accurate detection of the Wuhan virus is imperative to avoid false positives, since 2019-nCoV [2,3] shares significant homology [4] with other species.On the 24th of Jan, the CDC has published a list of 3 probes (Fig 1) for detecting the 2019-nCoV. Detection of all three probes would give a positive result, while detection of one or two gives an ’inconclusive result’ (and obviously negative when none are found).All 3 probes will have significant homology in other strains (Table 1 and Fig 2 ) - specifically SARS (Severe Acute Respiratory Syndrome). A much more specific option is to look within a 500bp genomic fragment in the N-terminal of the spike protein (Accid:NC 045512.2 [2],21852-22427, SI.cdc:nCoVFULLSLICE.fa), which seems to the most perturbed in this virulent strain [5].It is also important to have a common repository for all countries to be following a similar protocol. Many papers do no provide what PCR was used to detect coronaviruses [6].

Next EU summit may alter some of Turkish migrant deal

Significance Leaders have tried to put a positive spin on the inconclusive result, insisting they outlined the main principles of a deal and would work out details over the next ten days before convening again at the scheduled March 17-18 European Council summit to conclude an agreement. Impacts EU leaders are desperate to find a common approach to the refugee crisis before it leads to complete unravelling of the Schengen area. Such leaders as Germany's Merkel face huge political pressure to reduce the refugee influx which is fuelling support for extremist parties. If the plan works, it may lift the severe pressure on Greece by reducing the inflow of migrants and allowing relocation in the EU.